Therapeutic use of methionine to reduce the toxicity of platinum-containing anti-tumor compounds

ABSTRACT

Methods of preventing or reducing ototoxicity in patients undergoing treatment with therapeutically effective amounts of platinum-containing chemotherapeutic agents such as cisplatin or aminoglycoside antibiotics are provided. The methods comprise administering an effective amount of an otoprotective agent comprising methionine or a methionine-like moiety to said patient prior to, simultaneously with, or subsequently to administration of the platinum-containing chemotherapeutic agent or aminoglycoside antibiotic. Combinations of these time periods can also be employed.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of pending U.S. patentapplication Ser. No. 09/911,195 filed Jul. 23, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/057,065filed Apr. 8, 1998 (issued as U.S. Pat. No. 6,265,386), which is acontinuation-in-part of U.S. patent application Ser. No. 08/942,845filed Oct. 2, 1997 (issued as U.S. Pat. No. 6,187,817), which claimspriority from U.S. Provisional Application Ser. No. 60/027,750 filedOct. 3, 1996. The entire texts of U.S. patent application Ser. Nos.09/911,195, 09/057,065, 08/942,845, and U.S. Provisional ApplicationSer. No. 60/027,750 are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of protective agents in cancerchemotherapy in human and animal subjects. Protective agents arecompounds that prevent, reduce, or otherwise ameliorate the toxic sideeffects of anti-cancer chemotherapeutic compounds in normal body cellswhile substantially preserving the anti-tumor properties of thesecompounds in vivo when administered prior to, concomitantly with, orsubsequently to administration of such chemotherapeutic compounds. Morespecifically, the present invention relates to the use of D-methionineand structurally related compounds as protective agents havingoto-protective, weight loss-protective, gastrointestinal-protective,neuro-protective, alopecia-protective, and survival-enhancing effects inconjunction with chemotherapy employing platinum-containinganti-neoplastic agents, such as cisplatin. The present invention alsorelates to the use of D-methionine and structurally related compounds asprotective agents having oto-protective effects against noise-induced,loop diuretic-induced, aminoglycoside antibiotic-induced, ironchelator-induced, quinine- and quinidine-induced, and radiation-inducedhearing loss, as well as protective effects in ameliorating otherradiation-induced side effects such as neural damage, alopecia,gastrointestinal disorders, and reduced patient survival.

2. Description of Related Art

Cisplatin Chemotherapy

Cisplatin (cis-diamminedichloroplatinum(II); CDDP) is a widely usedantineoplastic agent. Cisplatin administration has increased both in thevariety of cancer types for which it is employed and in the amount usedin a given individual to achieve maximal therapeutic effect (Blumenreichet al., 1985; Forastiere et al., 1987; Gandara et al., 1989).

The toxic side effects of cisplatin have long been recognized and arewidely reported (Lippman et al., 1973; also see the review by Hacker,1991). These toxicities include a variety of peripheral neuropathies,myelo-suppression, gastrointestinal toxicity, nephrotoxicity, andototoxicity (Ozols and Young, 1985; Stewart et al., 1987; Stoter et al.,1989). Initially, the primary dose-limiting factor was nephrotoxicity,but now the routine administration of mannitol, hypertonic saline, andhigh fluid administration have ameliorated, but not eliminated, thatside effect. However, ototoxicity remains uncontrolled (Bajorin et al.,1987; Fillastre and Raguenez-Viotte, 1989). Although nephrotoxicity canstill be dose-limiting, currently the primary dose-limiting factor isototoxicity (Blumenreich et al., 1985; Forastiere et al., 1987; Berry etal., 1990).

The primary ototoxic effects of cisplatin appear to occur in thecochlea. Anatomical changes occur in both the stria vascularis and theorgan of Corti. The primary histologic findings include hair celldegeneration and damage to the supporting cells that are dose-related(Anniko and Sobin, 1986). At high doses, total collapse of themembranous labyrinth can occur (Anniko and Sobin, 1986). In the organ ofCorti, there is loss of outer and inner hair cells, with a propensityfor outer hair cell loss in the basal turn (Fleischman et al., 1975;Komune, 1981; Estrem et al., 1981; Schweitzer, 1993), and alterations inthe supporting cells and Reissner's membrane (Komune, 1981; Estrem etal., 1981). Estrem et al. (1981) also reported softening of thecuticular plate and an increased number of lysosomal bodies in theapical portion of the outer hair cell. However, the mechanisms inducingthese changes are largely unknown.

For equivalent inner ear concentrations, cisplatin is the most ototoxicdrug known (Moroso and Blair, 1983; Koegel, 1985; Anniko and Sobin,1986; Griffin, 1988). Generally, cisplatin ototoxicity is irreversible,its onset insidious, and the hearing loss may progress afterdiscontinuation of the protocol (Schaefer et al., 1985; Melamed et al.,1985; Pollera et al., 1988; Aguilar-Markulis et al., 1981; see thereview by Moroso and Blair, 1983). Hearing loss is usually permanent(Vermorken et al., 1983). Partial recovery may occur in some cases, butonly one of 121 patients with hearing loss had complete recovery in astudy by Aguilar-Markulis et al., (1981). Hearing loss typically startsat the ultra high frequencies (9000 to 20000 Hz) (Fausti et al., 1984;Kopelman et al., 1988) and then progresses into the high conventionalaudiometric range (Laurell and Engström, 1989; Kopelman et al., 1988;Meyer, 1989), reducing the patient's ability to hear consonant but notvowel sounds. An inability to understand speech and tinnitus arefrequent complaints (Kopelman et al., 1988). An increasing number ofpatients survive chemotherapy, but frequently with hearing impairment.

Nucleophilic Sulfur Protective Agents

Many sulfur-containing compounds (including substances with thio, thiol,and thioether groups) have been reported to provide CDDPnephroprotection in animal models (Anderson et al., 1990; Jones andBasinger, 1989; Jones et al., 1986; 1991a, b, c; 1992). These compoundsmay act by preventing the CDDP-induced depletion of glutathione or thebinding of CDDP to protein sulfhydryl groups (Hanneman and Baumann,1988; Nakano and Gemba, 1989; Gandara et al., 1989; Ravi et al., 1991;Schweitzer, 1993).

Additionally, sodium thiosulfate (STS) and diethyldithiocarbamate (DDTC)provide good CDDP otoprotection in animals (Otto et al., 1988; Church etal., 1995; Rybak et al., 1995). Unfortunately, STS may reduce CDDPtumoricidal action (Pfeifle et al., 1985; Aamdal et al., 1987) and mayexacerbate CDDP-induced weight loss and mortality (Otto et al., 1988).DDTC does not interfere with antitumor action (Qazi et al., 1988; Berryet al., 1989; Dedon et al., 1984; Borch et al., 1988), but can producesevere side effects (Rothenberg et al., 1988; Qazi et al., 1988).

D-Methionine

D-methionine (D-Met) is a sulfur-containing nucleophile that provideshighly effective CDDP nephroprotection in animals without decreasinganti-tumor action (Jones and Basinger, 1989). Although only tested inthat single study at a single dose level, D-Met was the most effectiveCDDP nephroprotectant that did not interfere with CDDP tumoricidalaction out of nearly 40 sulfur-containing agents tested in a series ofstudies by Jones and colleagues (Jones and Basinger, 1989; Jones et al.,1986; 1991a, b, c; 1992). As far as the inventor is aware, D-Met hasnever been previously tested as a CDDP otoprotectant, and has not yetbeen tested clinically (Treskes and van der Vijgh, 1993).

Sulfur-Containing Protective Agents and the Modulation ofCisplatin-Induced Toxicity

Studies indicate that individual sulfur-containing protective agents mayonly be effective in reducing specific types of toxicity, such asnephrotoxicity, while remaining ineffective in blocking otherplatinum-related complications such as peripheral neuropathy andototoxicity (Schweitzer, 1993). In addition, an agent which is effectiveas a regional chemoprotector following site-specific (intraperitoneal)usage of platinum-containing compounds such as CDDP may fail to provideadequate systemic protection, or may inhibit antitumor activity(Schweitzer, 1993).

Not all sulfur-containing compounds provide protection against all ofCDDP's toxicities, and it is not possible to predict which protectiveagents will be effective or ineffective for this purpose. For example,cefoxitin (Jones et al., 1992) does not provide nephroprotection.Ethyl-L-cysteinate and N-(2-mercapto- propionyl)glycine (Jones andBasinger, 1989) exacerbate CDDP nephrotoxicity. 2-(methylthio)nicotinicacid does not provide nephroprotection in rats (Jones et al., 1991b).The sodium salt of penicillin G does not protect against CDDPnephrotoxicity or weight loss (Jones et al., 1992). Similarly,thiamine-HCl does not protect against cisplatin nephrotoxicity or weightloss (Jones et al., 1992).

Furthermore, sulfur-containing compounds protective against one type ofCDDP toxicity frequently do not protect against other CDDP toxicities,and it is not possible to predict the specific antitoxic effectivenessof such compounds. Cephalexin (Jones et al., 1992) protects againstCDDP-induced kidney dysfunction and weight loss, but curiously does notprevent kidney pathology. Cefoxitin (Jones et al., 1992) provides someprotection against CDDP-induced weight loss, but does not protectagainst CDDP nephrotoxicity. The sodium salt of penicillin G does notprotect against either CDDP-induced nephrotoxicity or weight loss (Joneset al., 1992). Sulfathiazole provides protection against CDDPnephrotoxicity, but not weight loss (Jones et al., 1992).

WR2721 provides excellent CDDP nephroprotection (Mollman et al., 1988),but does not ameliorate nausea and vomiting (Glover et al., 1987). Nordoes WR2721 seem to provide CDDP otoprotection: Glover et al. (1987)found mild to severe hearing loss in 20 of 36 patients receiving WR2721prior to CDDP although nephroprotection was obtained. Rubin et al.(1995) reported a 45% incidence of significant hearing threshold shiftin patients pretreated with WR2721 prior to CDDP administration.Unfortunately, neither the Glover et al. (1987) nor Rubin et al. (1995)studies employed a control group, and both reported a high incidence ofototoxicity in patients receiving WR2721. In hamsters, Church et al.(1995) reported no WR2721 protection from ototoxicity or mortality.

Even when a sulfur-containing agent is found to be protective, its sideeffects can be so severe that clinical applicability is precluded. Inaddition, even among agents that provide CDDP otoprotection, theprotection may be so inconsistent and/or the side effects so great thatthey would not be used clinically. For example, DDTC provides protectionagainst CDDP-induced nephrotoxicity (Qazi et al., 1988; Berry et al.,1989; Gandara et al., 1989a, 1989b, and 1991) and ototoxicity (Church etal., 1995), but the protection against ototoxicity may only be partial(Gandara et al., 1989a; Ravi et al., 1992) and its side effects aresevere (Rothenberg et al., 1988; Berry et al., 1990). If DDTC dosing isreduced to ameliorate its side effects, adequate protection from CDDPside effects may not occur (Paredes et al., 1988). Similarly, disulfiram(Antabuse), which can be used as a precursor for its metabolite DDTC,can cause sensorimotor neuropathy (Argov and Mastiglia, 1979) andreversible confusion that can be dose-limiting (Stewart et al., 1987).Consequently, it is unlikely that DDTC will be widely used clinically asa CDDP chemoprotectant. In contrast, as described below, D-Met providescomplete otoprotection without apparent adverse side effects.

Finally, many sulfur-containing compounds inhibit the anti-tumor actionof CDDP, and it is not possible to predict which agents will or will notact in this manner. Thus, many agents that provide CDDP protection arenot clinically useful. For example, Captropril (Jones et al., 1992)protects against CDDP nephrotoxicity, but reacts immediately with CDDPto form a precipitate if coadministered, thereby precluding anti-tumorefficacy. L-methioninamide (Jones et al., 1991b) provides excellent CDDPnephroprotection, but impairs CDDP anti-tumor action. Metallothionein, asulfur-containing compound the synthesis of which is induced byadministration of bismuth subnitrate, provides CDDP nephroprotection,but also inhibits CDDP anti-tumor action (Naganuma et al., 1987;Boogaard et al., 1991; Satoh et al., 1993; Imura et al., 1992; Endresenet al., 1994). STS reduces CDDP nephrotoxicity (Pfeifle et al., 1985;Howell et al. 1982) and ototoxicity (Otto et al., 1988; Church et al.,1995), although some authors report inadequate otoprotection (Markman etal., 1985). However, STS will probably not be clinically useful ascoadministration with CDDP reduces the latter's tumoricidal action(Pfeile et al., 1985; Aamdal et al., 1987; Jones et al., 1991b), and tworoute administration does not provide nephroprotection (Jones et al.,1991b). Even in the absence of other agents, STS may also increasemortality and induce weight loss (Otto et al., 1988). Biotin, anothersulfur-containing compound that provides good CDDP nephroprotection,inhibits anti-tumor activity (Jones et al., 1992).

Thus, a variety of sulfur-containing compounds can act as protectiveagents for particular toxicities. A comparison of C—SH— and C—S—C—containing compounds demonstrated that the C—S—C— group was moreeffective in preventing nephrotoxicity in rats (Jones et al., 1989).However, not all of the compounds possessing the C—S—C— group were foundto be effective cisplatin antagonists.

The foregoing discussion demonstrates that it is not possible to predictreliably which particular sulfur-containing nucleophile will exhibit aplatinum-containing compound protective effect in any particular type ofcell, tissue, or organ. Indeed, individual compounds seem to exert theirprotective effects only in certain tissues. Thus, the ability of anyparticular nucleophilic sulfur compound to act as a protective agent inany particular tissue can only be determined by direct experimentation.Of course, such compound will only be of value if it does notsubstantially reduce the anti-tumor efficacy of cisplatin or relatedanti-tumor platinum-containing compounds.

Deegan et al. (1994) demonstrated that male Wistar rats receiving asingle intraperitoneal dose of cisplatin-methionine at a 1:5 ratio byweight did not exhibit cisplatin-induced nephrotoxicity. Their resultsindicated that cisplatin-methionine is significantly cytotoxic, yetlacks cisplatin-associated renal toxicity. These workers suggested arole for either methionine co-treatment or cisplatin-methioninecompounds in the treatment of human cancers. However, they neitherdisclosed nor suggested the specific otoprotective, weightloss-protective, gastrointestinal-protective, neuroprotective,alopecia-protective, or survival-enhancing effects of D-methioninesurprisingly discovered by the present inventor. Nor did they provideany motivation to investigate D-methionine as an otoprotectant, weightloss-protectant, survival-enhancing agent, etc., or any reasonableexpectation that methionine could act in these manners during cisplatinadministration. Finally, Deegan et al. provided no guidance orsuggestion as to how methionine could be used as a protective agent forvarious toxicities in humans, as described herein. As noted bySchweitzer (1993; page 12), while various nucleophilic sulfur protectiveagents have been shown to be effective in blocking or reversing therenal toxicity of CDDP while retaining the chemotherapeutic activity ofthe drug, each agent has to be considered individually. The effects onantineoplastic activity, individual CDDP toxicities, and appropriatedosing schedules need to be determined on a per se basis for eachcompound.

In view of the foregoing, the utility of D-Met as a highly effectiveplatinum-containing anti-neoplastic agent otoprotectant, weight lossprotectant, gastrointestinal protectant, neuroprotectant, alopeciaprotectant, and survival-enhancing agent which does not interfere withanti-tumor activity, and which does not appear to cause any serious sideeffects, could not have been predicted. In fact, the discovery ofD-Met's beneficial effects is surprising in view of the many significantproblems, discussed above, encountered with previously describedsulfur-containing nucleophiles that preclude their clinical use.

SUMMARY OF THE INVENTION

The present inventor has addressed the long-felt need in the art forprotective agents effective in preventing or ameliorating various toxiceffects of cisplatin and other platinum-containing anti-tumor compounds,but which do not significantly affect the antineoplastic activity ofthese compounds, and which do not themselves cause deleterious sideeffects as a result of their administration. She has also addressed thelong-felt need in the art for protective agents effective in preventingor ameliorating various toxic effects of aminoglycoside antibiotics,loop diuretics, iron chelating agents, quinine, quinidine, noise, andradiation. She has surprisingly discovered that D-methionine, andstructurally related compounds, can be used as an otoprotectant, aweight loss protectant, a gastrointestinal protectant, aneuroprotectant, an alopecia protectant, and a survival-enhancing agentduring treatment of a mammal with such compounds, or due to exposure tonoise or radiation.

Accordingly, in one aspect, the present invention provides a method forpreventing or reducing ototoxicity in a human or animal patientundergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-ototoxic effective amount of a methionineprotective agent. Such protective agent can be administered prior to,simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In another aspect, the present invention provides a method forpreventing or reducing weight loss in a human or animal patientundergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-weight loss effective amount of a methionineprotective agent. Such protective agent can be administered prior to,simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In another aspect, the present invention provides a method forpreventing or reducing gastrointestinal toxicity in a human or animalpatient undergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-gastrointestinal toxicity effective amount of amethionine protective agent. Such protective agent can be administeredprior to, simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In yet another aspect, the present invention provides a method forpreventing or reducing neurotoxicity in a human or animal patientundergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-neurotoxicity effective amount of a methionineprotective agent. Such protective agent can be administered prior to,simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In still another aspect, the present invention provides a method forpreventing or reducing alopecia in a human or animal patient undergoingtreatment with an anti-cancer effective amount of a platinum-containingchemotherapeutic agent, comprising administering to said patient ananti-alopecia effective amount of a methionine protective agent. Suchprotective agent can be administered prior to, simultaneously with, orsubsequently to administration of said platinum-containingchemotherapeutic agent. Combinations of these time periods can also beemployed.

In yet another aspect, the present invention provides a method forprolonging the survival of a human or animal patient undergoingtreatment with an anti-cancer effective amount of a platinum-containingchemotherapeutic agent, comprising administering to said patient asurvival-prolonging effective amount of a methionine protective agent.Such protective agent can be administered prior to, simultaneously with,or subsequently to administration of said platinum-containingchemotherapeutic agent. Combinations of these time periods can also beemployed.

In another aspect, the present invention provides a method forpreventing or treating ototoxicity in a patient undergoing treatmentwith an aminoglycoside antibiotic. The method comprises administering aneffective amount of an otoprotective agent comprising methionine or amethionine-like moiety to the patient.

In another aspect, the present invention provides a method forpreventing or reducing ototoxicity in a patient exposed to noise for atime and at an intensity sufficient to result in ototoxicity, comprisingadministering to the patient an anti-ototoxic effective amount of amethionine protective agent.

In yet another aspect, the present invention provides a method forpreventing or reducing ototoxicity, neurotoxicity, alopecia,gastrointestinal disorder, or reduced survival in a patient exposed toradiation for a time and at an intensity sufficient to result inototoxicity, neurotoxicity, alopecia, gastrointestinal disorder, orreduced survival, comprising administering to the patient an effectiveamount of a methionine protective agent.

The present invention also provides compositions, includingpharmaceutical compositions, comprising the present methionineprotective agents, either alone, or in combination with aplatinum-containing chemotherapeutic agent, an aminoglycosideantibiotic, a loop diuretic agent, an iron chelating agent, quinine, orquinidine.

Such protective agents can be administered prior to, simultaneouslywith, or subsequently to administration of the platinum-containingchemotherapeutic agent, the aminoglycoside antibiotic, loop diureticagent, iron chelating agent, quinine, quinidine, or exposure to a toxiclevel of noise or radiation. Combinations of these time periods can alsobe employed.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the following detailed descriptionand examples, while indicating preferred embodiments of the invention,are given by way of illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 shows ABR post-test thresholds (means ±1 S.D.) for the variousanimal groups for all stimuli including: a) clicks; b) 1000 Hztonebursts; c) 4000 Hz tonebursts; d) 8000 Hz tonebursts; and e) 14000Hz tonebursts. * indicates significantly different from the CDDP-treatedcontrols at the p≦.01 level.

FIGS. 2A-2F are SEM photomicrographs of: A) middle turn of untreatedcontrol; B) middle turn of treated control (16 mg/kg CDDP); C) middleturn of animal administered 300 mg/kg D-Met prior to the 16 mg/kg CDDPdose; D) basal turn of untreated control; E) basal turn of treatedcontrol (16 mg/kg CDDP); and F) basal turn of animal administered 300mg/kg D-Met prior to the 16 mg/kg CDDP dose.

FIG. 3 shows the average weight loss in grams for the various animalgroups. * indicates significantly different from the CDDP-treatedcontrols at the p≦.01 level.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. Even so, this detaileddescription should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

The contents of each of the references cited herein are hereinincorporated by reference in their entirety.

EXAMPLE 1 Otoprotective Effect of D-Met

This experiment demonstrates the effectiveness of D-Met in preventing avariety of different toxic side effects associated with the use ofplatinum-containing anti-tumor compounds, exemplified by CDDP(cisplatin), in a mammal.

Materials and Methods

Animals

As is well known to those of ordinary skill in the art, the rat is awell-accepted experimental animal useful as a model for studies of CDDPtoxicity in humans.

Complete data sets were obtained for five groups of five male Wistarrats (280-421 g). All animals were anesthetized with 1 ml/mg IM ofRompun cocktail (a solution containing 86.21 mg/ml ketamine and 2.76mg/ml xylazine) prior to all injections and testing. Anesthesia wassupplemented as needed with half doses throughout testing. The fivegroups included: a treated control group which received 16 mg/kg CDDPdissolved in normal sterile saline (1 mg of CDDP/ml normal saline;solution pH 6.3) administered by i.p. infusion with a Harvard ApparatusInfusion Pump, over a 30 minute period, an untreated control group thatreceived an equivalent volume of normal saline (pH 6.5) instead of CDDP,and three experimental groups that received either 75, 150, or 300 mg/kgD-Met dissolved in 3-5 ml of normal saline (solution pH 6.6) deliveredby slow (over 1-2 minutes) i.p. injection 30 minutes prior to the sameCDDP infusion as the treated control group. Both CDDP (purchased fromSigma Chemical Co., St. Louis) and D-Met (purchased from Acros Organics,Pittsburgh, Pa.) were freshly prepared before each experiment. For thetreated control group, a total of 10 animals were needed to obtain 5animals with complete data sets because 50% of the animals did notsurvive to the end of the study period. Only 5 animals were needed inthe untreated control and in each of the D-Met pretreated groups becauseall of the animals in each of those groups survived until the end of thestudy period.

All of the care and use of the animals was approved by the SouthernIllinois University School of Medicine Laboratory Animal Care and UseCommittee, and was under the supervision of the Southern IllinoisUniversity School of Medicine Unit for Laboratory Animal Medicine.

Evoked Potentials

Auditory Brainstem Testing (ABR) was used to assess auditory threshold.Testing occurred just prior to administration of the CDDP or saline(with or without a protective agent) and again 3 days later. All testingwas performed with the animal in a double walled IAC booth.

Platinum/iridium needle electrodes were placed at the vertex(non-inverting) to a point directly below the ipsilateral pinna(inverting) with a ground electrode placed in the hind leg.

ABR data collection was obtained with a Biologic Traveler system with anadditional custom made high frequency stimulator for 14000 Hz. ABRthresholds were measured in response to 100 microsecond clicks and fortonebursts with 1 ms rise/fall and 0 ms plateau gated by a Blackmanenvelope and centered at the frequencies of 1, 4, 8, and 14 kHzpresented at 10/s. An intensity series was obtained for each animal from100 to 0 dB peak equivalent SPL (peSPL) for click stimuli and SoundPressure Level (SPL) for tonebursts in 10 dB decrements. The term peSPLmeans that the amplitude of the click stimulus from the prestimulusbaseline to the first peak is equivalent to the SPL of a pure tonestimulus having the same prestimulus baseline to peak amplitude.Threshold was defined as the lowest intensity capable of eliciting areplicable, visually detectable response.

A total of 512 sweeps constituted each average. The recording epoch was15 ms following stimulus onset. Responses were analogue filtered with a30-3000 Hz bandpass.

Rectal temperature was monitored throughout recordings, with animaltemperature being maintained by a warming pad.

Electron Microscopy

The animals were sacrificed by decapitation while under generalanesthesia and cochleae perfused with fixative through the perilymphaticspaces. The primary fixative was 2.5% glutaraldehyde at 4° C. in 0.1Mphosphate buffer (pH 7.4). A small hole in the otic capsule was handdrilled beneath the first turn with a three sided, sharpened pick. Invitro perfusion was performed intermittently within 5 minutes ofsacrifice through the small hole in scala tympani, allowing the fluid toexit through the opened oval window. After perfusion fixation, the roundwindow membrane was removed, and the cochleae were immersed inglutaraldehyde and stored in the refrigerator overnight.

After overnight fixation in glutaraldehyde, the cochleae were rinsed in0.1 M phosphate buffer and gently perfused with the buffer through theperilymphatic spaces by loosely fitting the tube end of the perfusionsyringe over the opening drilled in the scala tympani. Cochleae werethen rinsed in buffer 3 times. After rinsing, the cochleae werepost-fixed by a perfusion of 1.5% OsO4 (at 4° C.) in phosphate buffer ina fume hood. Fixation was continued by immersion and rotation in thesame fixative for 15 minutes. The cochleae were rinsed in the samefashion as after glutaraldehyde fixation.

Under the dissecting microscope, the bony capsule of the cochlea wascarefully removed.

The tissue was then serially dehydrated in 2×50%, 70%, 85%, 95% and3×100% ethanol. Each specimen was dried using Peldri and placed on astub for sputter coating with 13 nm platinum. The tissue was viewedthrough a Hitachi S-500 scanning electron microscope and photographstaken on Polaroid type 55 land Film.

Semi-quantitative analysis per turn for the outer hair cells wasperformed in the following manner: For each turn of the cochlea, apical,middle, and base, a representative sample was examined. For each sample,11 inner hair cells served as a guide to count a section of 33 outerhair cells or 11 per row. The number of damaged or missing outer haircells within each sample was then counted.

Weight

Each animal's weight was measured in an Ohaus triple beam balance scalebefore administration of the anesthetic for the pretest and again beforethe post-test 3 days later.

Statistical Analysis

ABR data were analyzed using a three factor analysis of variance (ANOVA)with one between subject factor (groups) and two within subject factors(frequency and pre- vs. post-test). Each dependent variable was analyzedindependently. Tests subsequent to the ANOVA were carried out inaccordance with the Tukey HSD procedure. Weight loss and/orgastrointestinal protection was measured using the same type ofstatistical analysis as the ABR measures. SEM data were analyzed foreach turn using a one way analysis of variance with Post-Hoc Tukey HSDanalysis. The criterion for statistical significance for all measureswas p≦0.01.

Results

Hearing Loss

Post test ABR hearing thresholds are presented in FIG. 1. As expected,no significant threshold shift in response to any stimulus occurred inthe untreated control group, and marked significant threshold shiftoccurred in response to all stimuli, but particularly for the highfrequencies, in the treated control group. For the animals receivingD-Met prior to the CDDP, 2/5 and 3/5 animals receiving 75 and 150 mg/kgD-Met, respectively, had complete otoprotection as defined by nosignificant ABR threshold shift for any stimulus. For the 300 mg/kgD-Met administration, all 5 animals had complete otoprotection for allstimulus conditions (FIG. 1). All experimental groups receiving anylevel of D-Met had significantly lower ABR thresholds than the treatedcontrol group for all stimuli, as did the untreated control group. Thisobserved protection from hearing loss may occur not only as a result ofprotection of cochlear mechanisms, but also as a result of protection ofthe auditory neural pathway (i.e., neuroprotection).

Histology

Histologic findings (FIG. 2) were consistent with the ABR findings. Allgroups had essentially normal hair cell counts for the apical turn, withno significant difference between groups. For the middle and basalturns, only the treated control group showed significantly differentfindings from the untreated control group and from the three groupsreceiving preadministration of D-Met, with the basal turn beingconsistently more affected than the middle turn.

Weight Loss

CDDP-induced weight loss diminished as D-Met dosing increased (FIG. 3).Weight loss in the experimental group receiving 300 mg/kg wassignificantly less than that in the treated control group. The amount ofweight loss across groups was significantly correlated with the amountof threshold shift for all stimuli, with the highest correlation for the14 kHz stimulus.

Neuroprotection

Animals receiving D-Met were noticeably more lively, active, andcoordinated on the morning of the third day as compared to the survivingtreated control group animals.

Alopecia

The coats of animals receiving D-Met were noticeably superior to thoseof control group animals, and showed significantly less hair loss.

Survival During the Study Period

All 15/15 animals receiving any level of D-Met survived to the end ofthe study period as compared to 5/10 treated control group animals.

Discussion

The foregoing results demonstrate that 300 mg/kg D-Met administered 30minutes before 16 mg/kg CDDP provides complete otoprotection, asindicated by ABR and histologic findings, while also reducingCDDP-induced weight loss, gastrointestinal toxicity, neurotoxicity,alopecia, and improving survival.

While not intending to be bound to any particular theory, I hypothesizethat D-Met may provide these protective effects by any one or more of anumber of different mechanisms.

According to Schweitzer, (1993), sulfur-containing compounds may preventCDDP from interacting with intracellular target molecules, thenucleophilic oxygen or sulfur atoms interacting with the electrophilicsite of the CDDP, thus displacing or extracting platinum after it isbound. Theoretically, these agents provide protection because of theirhigh affinity for platinum complexes. It is known that CDDP reacts withmethionine's sulfhydryl group (Lempers and Reedijk, 1990).

CDDP may preferentially bind to free D-Met, thus protecting glutathione.Reduced glutathione is an essential part of the anti-oxidant pathways.CDDP does reduce renal glutathione levels, resulting in increased lipidperoxidation (Hanneman and Baumann, 1988; Sugihara et al., 1987a, b;Boogaard, 1991). CDDP also reduces glutathione levels in the cochlea andinferior colliculus (Ravi et al., 1991). More recent work (Ravi et al.,1995, Rybak et al., 1995) investigated changes specifically in thecochlear antioxidant system. Systemic CDDP administration decreasedreduced glutathione (GSH) levels, and reduced activity of the enzymesglutathione peroxidase (GSH-Px) and glutathione reductase (GR). Oxidizedglutathione or glutathione disulfide (GSSG) was not found, suggestingthat the overall glutathione levels decreased rather than merely beingoxidized. Ravi et al., (1995) also reported increased cochlearmalondialdehyde (MDA) levels, reflecting increased lipid peroxidation.Because CDDP does increase the level of free radicals in general(Hanneman and Baumann, 1988), preservation of the anti-oxidant systemmay be critical in preventing CDDP side effects.

D-Met preadministration may protect the sulfur groups of proteins,including protein bound L-methionine. CDDP binds to the methioninegroups in protein and to glutathione (Lempers and Reedijk, 1990).Schweitzer, (1993) suggests that platinum binding to protein sulfhydrylgroups may cause CDDP nephrotoxicity, accounting for thenephroprotective action of thiols (Gandara et al., 1989). It is logicalthat free D-Met may preferentially bind to CDDP because of the sterichindrance of the protein bound sulfur groups. This protection couldoccur by preferential binding of the CDDP to D-Met, or perhaps D-Metcould reverse the Pt binding to the protein-bound methionine andglutathione, as do other sulfur-containing compounds (Lempers andReedijk, 1990). Methionine can displace plasma-bound Pt (Alden andRepta, 1984).

D-Met binding to CDDP may also protect free L-methionine (L-Met), anessential amino acid. Parenteral administration of DL-methionine inhumans results in higher plasma levels of the D- isomer (Printen et al.,1979). Because the D-Met is less well metabolized than L-Met in humans,it may remain more available for CDDP binding, thus protecting the L-Metfor needed protein synthesis, cell activation, and metabolism.

Fortunately, D-Met does not inhibit CDDP anti-tumor action as determinedagainst the Walker 256 carcinosarcoma in the rat (Jones and Basinger,1989). Preadministration of methionine, presumably a racemic mixture,actually sensitized NHIK 3025 in vitro human uterine cervix carcinoma insitu cancer cells to CDDP cytotoxicity (Melvik and Petterson, 1987).

Several factors may account for D-Met's CDDP-protective action innontumor cells as compared to tumor cells. Methionine metabolism isclearly different in tumor and nontumor cells (Hoffman, 1985), but howthese differences may result in differential CDDP action has not beenelucidated. The toxic effects of CDDP may also be different in tumor andnontumor cells. The CDDP anti-tumor effect results primarily fromcisplatin's reaction with DNA, primarily at the N-7 bisguanine position.Initially, mono-adducts are formed, followed by rapid intra-strandcross-linking, causing cytotoxicity (see the review by Tognella, 1990).The binding of platinum to cytosolic ligands and nucleoprotein fractionsmay also play a role, but the receptors and interactions are not yetdefined (Schweitzer, 1993). Significant DNA binding in normal cells isless likely because fewer DNA replication forks are open at any point intime, unlike in rapidly dividing tumor cells. In nontumor cells, thetoxic effects may be largely secondary to the binding with amino acids,either free or protein-bound, and deactivation of the antioxidantpathway, as described above.

The timing of CDDP reactions may also be different in tumor and nontumorcells. CDDP uptake by the Walker 256 carcinosarcoma in the rat is veryrapid, occurring in the first few minutes after administration, followedby a rapid redistribution that is complete within 15 minutes afterinjection (Jones and Basinger, 1989). Because the uptake of CDDP intotumor cells is very rapid, the binding to the DNA bisguanine groups,particularly at the open replication forks, may occur more rapidly thanthe reaction of CDDP with methionine.

Although CDDP uptake into the kidney is also rapid (Jones and Basinger,1989), CDDP binding to protein is relatively slow. As reviewed bySchweitzer, (1993), following IV cisplatin administration, 90% ofcisplatin is protein-bound within 2 hours, with half-lives of 25 to 50minutes and 53 to 73 hours for unbound and bound platinum, respectively.Platinum tissue levels decline slowly. Platinum may still be measuredover a week after high dosage administration, and bound fragments maystill be present when the patient starts the next treatment cycle.Platinum uptake in the stria vascularis and the organ of Corti increasesat least over a 24 hour period, which may underlie the dose-relatedcumulative ototoxicity (Schweitzer, 1993), but may also allow time forCDDP binding to D-Met before uptake into the cochlea.

However, the CDDP toxicities both in tumor and nontumor cells arecomplex, and many factors may be involved in D-Met's protective action.

A positive correlation between weight loss and outer hair cell loss inguinea pigs has been demonstrated (Tange et al., 1982, Hoeve et al.,1988), but both studies noted marked intersubject variability. The datapresented above reveal a positive correlation between weight loss andthreshold loss that increased as stimulus frequency increased. Thesignificant reduction in weight loss with 300 mg/kg D-Metpreadministration suggests that D-Met also alleviates some of thegastrointestinal toxicities of CDDP. The amelioration in weight loss byD-Met could also be related to a decrease in nephrotoxicity or otherfactors.

The elimination of CDDP mortality in this study by preadministration ofany of the three D-Met levels demonstrates a marked improvement in theoverall health status of the animals. D-Met preadministration maytherefore be useful in shifting the LD₅₀ level of CDDP and otherplatinum-containing anti-tumor agents, permitting the safe use of higherlevels of these agents during chemotherapy, with potential improvementof the cancer cure rate.

Therapeutic Applications

The data presented above demonstrate that D-Met prevents CDDP-inducedototoxicity, reduces CDDP-induced weight loss, protects againstCDDP-induced gastrointestinal toxicity, neurotoxicity, and alopecia, andimproves survival during CDDP treatment in a mammal. BecauseCDDP-induced hearing loss in humans is almost invariably permanent,prevention of this hearing loss has a number of important consequences.If ototoxicity could be prevented, not only could hearing be spared, butperhaps higher doses of cisplatin and other platinum-containingantineoplastic agents could be routinely employed during chemotherapy,increasing the effectiveness of anti-tumor therapy in human patients.

The foregoing data also suggest that D-Met and the other methionineprotective agents disclosed herein will be effective in the treatment ofnoise-induced, loop diuretic-induced, aminoglycoside antibiotic-induced,iron chelator-induced, quinine- and quinidine-induced, andradiation-induced ototoxicity, as well as in ameliorating otherradiation-induced side effects such as neural damage, alopecia,gastrointestinal disorders, and in improving patient survival.

As used herein, the term “ototoxicity” includes, but is not limited to,any detrimental or pathologic change in the structure or function of theear, including changes in hearing and balance. Auditory functionalchanges can include, but are not limited to, hearing loss or otherchanges in auditory threshold for any stimulus, perception of soundincluding recruitment (abnormal growth in the perception of loudness),ability to identify, localize, recognize, distinguish between, orprocess sounds, and/or distortion of sounds or any abnormality asmeasured by conventional auditory tests. This term also includestinnitus (ringing or noises in the ear), which includes any perceptionof sound that is not in response to an external signal. Further,ototoxicity includes any perceived or measured functional change in thebalance or vestibular system, including, but not limited to, eitherinduced or spontaneous vertigo, dysequilibrium, increased susceptibilityto motion sickness, nausea, vomiting, nystagmus, syncope,lightheadedness, dizziness, difficulty in visual tracking secondary tovestibular or balance disorder or abnormality as measured on any test ofvestibular or balance function. Structural changes can include anyintra- or extra-cellular, multicellular, or organ change in the auditoryor vestibular pathways from the external ear up through and includingthe cortex and all pathways in between.

The term “otoprotective agent” refers to an agent that prevents,ameliorates, or otherwise protects against ototoxicity.

The term “neurotoxicity” includes, but is not limited to, anydetrimental or pathologic change in the structure or function in theneurologic system or any part thereof. Neurologic functional changes caninclude, but are not limited to, neuropathy, either central or distal,including a common “stocking and glove” pattern, tingling, loss ofsensation, numbness, decreased vibratory sensation, decreased deeptendon reflexes, sensory ataxia, neuritis, focal encephalopathy,aphasia, autonomic neuropathy, postural hypotension, a myasthenia-likesyndrome, muscle cramps, headache, seizures, blindness or visualdisturbance secondary to disorder of the optic or visual neurologicalpathway, papilledema, hearing loss secondary to disorder of the auditoryneurologic pathway, and/or loss of the sensation of taste. Structuralchanges can include intra- or extra-cellular, multicellular, or organchanges, anywhere in the neurologic system, including both peripheraland central systems. Neurotoxicity can manifest itself during or afterthe course of treatment with platinum-containing anti-tumor compounds.

The term “neuroprotective agent” refers to an agent that prevents,ameliorates, or otherwise protects against neurotoxicity.

The term “gastrointestinal toxicity” includes, but is not limited to,any detrimental or pathologic change in the structure or function in thegastrointestinal system or any part thereof. Gastrointestinal changesinclude, for example, current or delayed nausea, vomiting, esophagealreflux, stomatitis, bleeding along the gastrointestinal tract, diarrhea,weight loss, and/or anorexia. Gastrointestinal toxicity can manifestitself during or after the course of treatment with platinum-containinganti-tumor compounds.

The term “gastrointestinal-protective agent” refers to an agent thatprevents, ameliorates, or otherwise protects against gastrointestinaltoxicity.

In view of the results presented above, the medical or veterinarypractitioner, by employing the compounds, compositions, and methodsdescribed below, will be able to maintain any of the foregoingparameters in a mammal, especially a human, at a level of from about 70%to about 80% of the pre-chemotherapy or other treatment or exposurelevel, more preferably from about 80% to about 90% of thepre-chemotherapy or other treatment or exposure level, most preferablyfrom about 90% to about 100% of the pre-chemotherapy or other treatmentor exposure level, as measured by standard tests routinely employed inthe art. These compounds and methods can also be used for the treatmentof domestic pets, such as cats and dogs.

The teachings presented herein permit the design of therapeutic regimensthat can be employed to reduce the undesirable side effects ofplatinum-containing anti-tumor compounds such as CDDP, increase thedosing of such anti-tumor compounds to obtain a higher cancer cure rate,and perhaps include weaker patients in treatment protocols employingsuch anti-tumor compounds, from which they are currently excludedbecause they cannot withstand the toxicities associated therewith. Thepresently disclosed teachings also permit the design of therapeuticregimens useful in preventing or reducing the undesirable ototoxic sideeffects of noise, loop diuretics, aminoglycoside antibiotics, ironchelators, quinine, quinidine, and radiation, as well as otherradiation-induced side effects such as neural damage, alopecia,gastrointestinal disorders, and decreased patient survival.

Administration of D-Met before, during, or after administration ofantineoplastic effective amounts of platinum-containing anti-tumorcompounds such as CDDP, or during various combinations of these timeperiods, is particularly useful in view of D-Met's lack of interferencewith CDDP anti-tumor action (Jones and Basinger, 1989; Melvik andPetterson, 1987).

D-Met and structurally related compounds can be used in conjunction withplatinum-containing antitumor compounds such as CDDP duringchemotherapy, and in conjunction with the use of loop diuretics,aminoglycoside antibiotics, iron chelators, quinine, and quinidine, asdescribed below. These methionine protective agents can also be used toprevent or reduce the ototoxic effects of noise and radiation, as wellas other radiation side effects, as described below as well.

Methionine and Its Derivatives

D-Met has been administered to humans for various purposes. For example,C-labeled D-Met has been used for radiographic imaging (Meyer et al.,1985), and DL-methionine has been administered for parenteral nutrition(Printen et al., 1979). D-Met has also been safely administered tohumans orally for nutritional studies (Kaji et al., 1987; Kies et al.,1975; Stegink et al., 1986). Oral methionine is sold as an over thecounter preparation to control urinary pH (Drug Facts and Comparisons,1991). The contraindications are for patients with a history of liverdisease, and that high dosage methionine may inhibit growth in childrenwhen given for an extended time period.

Analogs or derivatives of methionine useful in the present invention arecompounds containing a methionine moiety, or a methionine-like moietyincluding a thioether group, that exhibit an otoprotectant effect, aweight-loss protectant effect, a gastrointestinal protectant effect, aneuroprotectant effect, an alopecia protectant effect, and/or asurvival-enhancing effect when used in conjunction with an antitumorplatinum coordination compound administered in an effectivechemotherapeutic dose, a loop diuretic compound, an aminoglycosideantibiotic, an iron chelator, quinine, or in conjunction with exposureto noise or radiation. Among the compounds structurally related to D-Metthat can be employed in the present invention are those containing theC—S—C— (thioether) moiety. These include, but are not limited to,compounds having the structural formula:

wherein m is an integer from 0 to 3; n is an integer from 1 to 3;X=—OR¹, —OCOR¹, —COOR¹, —CHO, —CH(OR¹)₂, or —CH₂OH; Y=—NR²R³ or —OH;R¹=H or a substituted or unsubstituted, straight, branched chain, orcyclic alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms; R²=H or a substituted or unsubstituted, straight or branchedchain acyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms; and R³=H or a substituted or unsubstituted, straight or branchedchain acyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms; or a pharmaceutically acceptable salt thereof.

The lower alkyl and acyl groups described herein, either alone orcontaining the various substituents defined herein, can contain-from oneto six carbon atoms in the principal chain, and up to about 15 carbonatoms total. The lower alkyl groups include, for example, methyl, ethyl,propyl, isopropyl, butyl, hexyl, cyclopropyl, cyclopentyl, cyclohexyl,and the like. Substituents of the substituted alkyl and acyl groupsdescribed herein can include, for example, groups selected from alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, O, S, N, P, or halogen(Cl, F, Br, or I) atoms. Optionally, these substituent alkyl,cycloalkyl, etc., groups can be substituted with O, S, N, P, or halogen(Cl, F, Br, or I) atoms. These substituent alkyl, cycloalkyl, etc.,groups include, for example, lower alkoxy groups such as methoxy,ethoxy, and butoxy, and groups such as halo, nitro, amino, and keto.

The alkenyl groups described herein, either alone or with the varioussubstituents defined herein, are preferably lower alkenyl containingfrom two to six carbon atoms in the principal chain, and up to about 15carbon atoms total. They can be substituted, straight, or branchedchain, and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl,hexenyl, and the like.

The alkynyl groups described herein, either alone or with the varioussubstituents defined herein, are preferably lower alkynyl containingfrom two to six carbon atoms in the principal chain, and up to about 15carbon atoms total. They can be substituted, straight or branched chain,and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and thelike.

The aryl moieties described herein, either alone or with varioussubstituents defined herein, can contain from about 6 to about 15 carbonatoms, and include phenyl. Substituents include alkanoxy, protectedhydroxy, halogen, alkyl, aryl, alkenyl, acyl, acyloxy, nitro, amino,amido, etc. Phenyl is a preferred aryl.

The heteroaryl moieties described herein, either alone or with varioussubstituents defined herein, can contain from about 5 to about 15 atoms,and include, furyl, thienyl, pyridyl and the like. Substituents includealkanoxy, protected hydroxy, halogen, alkyl, aryl, alkenyl, acyl,acyloxy, nitro, amino, and amido.

The acyloxy groups described herein can contain alkyl, cycloalkyl,alkenyl, alkynyl, aryl, or heteroaryl groups.

The carbon atoms, i.e., the methyl and methylene groups, constitutingthe principal backbone of the methionine or methionine-like moiety canalso be substituted as variously described above.

Non-limiting examples of such methionine protective agents includeD-methionine (D-Met), L-methionine, a mixture of D-methionine andL-methionine, normethionine, homomethionine, methioninol, hydroxymethionine, ethionine, or pharmaceutically acceptable salts thereof.S-adenosyl-L-methionine, or a pharmaceutically acceptable salt thereof,can also be employed. Methionine protective agents of the presentinvention can be in the D-, L-, or DL- form, and includepharmaceutically acceptable N- (mono- and dicarboxylic acid) acylderivatives and alkyl esters thereof. Exemplary acyl derivatives includethe formyl, acetyl, propionyl, and succinyl derivatives. Exemplary esterderivatives include the methyl, ethyl, propyl, isopropyl, and butylesters. D-Met is a preferred compound.

Collectively, methionine, along with the other compounds discussedabove, can be referred to as “methionine protective agents.” Thesecompounds can be used alone or in various combinations with one anotherin the methods described herein.

These compounds can be administered alone, or in combination with theother drug compounds discussed herein, in the form of the water-solubleacid, free base, or as physiologically acceptable salts, including acidaddition salts formed with organic and inorganic acids, for example,hydrochlorides, hydrobromides, sulfates, phosphates, citrates,fumarates, and maleates, and cations such as sodium, potassium, etc.These compounds can be formulated for administration to humans andanimals with pharmaceutically acceptable carriers, excipients, anddiluents, such as sterile distilled water, Ringer's solution, normalsaline, 5% glucose, dextrose, fructose, sucrose, etc., and mixturesthereof, as is well known in the art. Antimicrobial agents,preservatives, etc., can also be included. Compositions for oraladministration can include coloring and flavoring agents. Additionalmethods of formulating compounds of the present invention foradministration in the methods described herein can be found, forexample, in Remington's Pharmaceutical Sciences, Fifteenth Edition, MackPublishing Company, Easton, Pa., 1975.

Anti-tumor Platinum Compounds

Cisplatin (CDDP; cis-diamminedichloro-platinum(II)) is currently theanti-tumor platinum coordination compound most frequently employed inthe therapy of testicular cancer, ovarian tumors, and a variety of othercancers. Methods of employing CDDP clinically are well known in the art(Nicolini, 1987). For example, CDDP can be administered in a single dayover a six hour period, once per month, by slow intravenous infusion.For localized lesions, CDDP can be administered by local injection.Intraperitoneal infusion can also be employed. CDDP can be administeredin doses as low as 10 mg/m² per treatment if part of a multi-drugregimen, or if the patient has an adverse reaction to higher dosing. Atthe low end, a more common clinical dose is about 30 mg/m²; the high endof the range is about 120 to about 150 mg/m² per treatment. When used inconjunction with D-Met or other methionine protective agents, thesedosages can be increased.

CDDP is representative of a broad class of water-soluble, platinumcoordination compounds well known in the art that provide platinum inthe form of an ion having anti-tumor activity. Among the anti-tumorplatinum coordination compounds described in the literature which areuseful in the methods of the present invention are, for example,trans-diaminedichloro-platinum(II), cis-diamine-diaquaplatinum(II)-ion,cis-diaminedichloroplatinum(II)-ion,chloro(diethylenetriamine)-platinum(II) chloride,dichloro(ethylenediamine)-platinum(II),diammine(1,1-cyclobutanedicarboxylato)-platinum(II) (carboplatin),spiroplatin, dichlorotrans-dihydroxybisisopropolamine platinum IV(iproplatin), diammine(2-ethylmalonato)-platinum(II),ethylenediamine-malonatoplatinum(II),aqua(1,2-diaminodiclohexane)-sulfatoplatinum(II),(1,2-diaminocyclohexane)malonato-platinum(II),(4-carboxyphthalato)(1,2-diaminocyclo-hexane)-platinum(II),(1,2-diaminocyclohexane)-(isocitrato)platinum(II),(1,2-diaminocyclohexane)-cis(pyruvato)platinum(II), and(1,2-diaminocyclohexane)-oxalatoplatinum(II).

Loop Diuretics

Loop diuretics are a group of compounds with dissimilar chemicalstructure, but which share a similar mechanism and site of action withinthe kidney: these compounds inhibit sodium chloride reabsorption at thehigh-capacity site in the thick ascending limb of the loop of Henle,causing greatly increased excretion of sodium chloride in the urine, andto a lesser extent of potassium. Loop diuretics are among the compoundsexhibiting the greatest diuretic effect, and are commonly used in thetreatment of edema of cardiac, hepatic, or renal origin. Use of thesecompounds can cause ototoxicity at least in part as a result of thealteration of electrolyte composition in the inner ear. In adults,ototoxicity is generally reversible, disappearing upon withdrawal of thedrug; however, permanent hearing loss has been reported, particularlywith ethacrynic acid. In neonates, reversibility of hearing loss may bepermanent. Commonly used loop diuretics include, but are not limited to,furosemide (Lasix and other compounds), ethacrynic acid (Edecrin),bumetanide (Bumex and other compounds), piretanide, muzolimine,indapamide (Lozol), and xipamide.

Loop diuretics greatly exacerbate the ototoxicity of platinum-containingantitumor compounds and aminoglycoside antibiotics. The interaction ofthese ototoxic compounds is synergistic; D-met and the other methionineprotective agents disclosed herein may prevent their ototoxicity orototoxic synergistic interaction.

Aminoglycoside Antibiotics

The aminoglycoside antibiotics share several structural features: theyeach contain one or more sugar moieties and a streptidine ring, and theyeach have one or more amino or guanidino groups. The currently availableaminoglycoside antibiotics include streptomycin, kanamycin, gentamicin,amikacin, neomycin, netilmicin, paromomycin, vancomycin, hygromycin,erythromycin and tobramycin. One of the principal dangers associatedwith the use of aminoglycoside antibiotics is their ototoxicity, whichis associated with either hearing loss (cochlear damage), vertigo(vestibular damage), or both. An early sign is tinnitus accompanied byloss of high-frequency hearing. Early detection of hearing loss can bereversed; prolonged treatment results in permanent hearing loss. Theconcomitant administration of other drugs that cause similar adverseeffects potentiates the adverse effects of the aminoglycosides. Suchother drugs include loop diuretic agents, as discussed above.

Iron Chelating Agents

Iron chelating agents such as deferoxamine mesylate (desferrioxaminemesylate; for example, Desferal) are used to treat patients exhibitingelevated levels of iron in the blood. Such patients include thosesuffering from sickle cell anemia, hereditary disorders resulting inelevated blood iron levels, those receiving frequent blood transfusions,those who have ingested large amounts of ferrous salts of iron (ironpoisoning), etc. The use of iron chelating agents such as deferoxaminecan result in ototoxicity.

Quinine and Quinidine

Quinine (Quinamm) has long been used as an antipyretic, analgesic, andantimalarial. Recently, it has been used to stabilize muscle membranesagainst repetitive activity. Specifically, it is used to treat myotoniacongenita (Thomsen's disease) and nocturnal muscle cramp. Quinidine is aclass IA antiarrhythmic agent useful in the treatment of atrial andventricular arrhythmias.

The adverse side effects of quinine and quinidine are similar, and havebeen given the name “cinchonism,” deriving from the fact that quinine isobtained from the bark of the cinchona tree. These side effects includedisturbances of hearing, including tinnitus, deafness, and vertigo.

Noise

Noise-induced hearing loss, both impulse and chronic exposure, candamage hearing. In impulse noise, including blast exposure, the patientmay suffer significant tympanic membrane and middle ear damage. Inchronic exposure, which generally occurs at lower intensity levels,middle ear and tympanic membrane damage are unlikely. In noise exposure,the primary and initial damage is generally cochlear, with secondaryneural degeneration of the auditory system occurring over time.Noise-induced hearing loss has been reviewed by the present inventor inthe book entitled Essential Audiology For Physicians (1998) SingularPublishing Group, Inc., San Diego.

Noise-induced hearing loss can occur secondary to a single very loudnoise exposure, or secondary to relatively high-level noise exposureover a long period of time. The risk of noise-induced hearing loss isrelated to both sound intensity and duration. Both the OccupationalSafety and Health Administration (OSHA) and the Environmental ProtectionAgency (EPA) have established standards relevant to noise exposurelevels in industry. The OSHA Permissible Noise Exposure Levels rangefrom a duration of 32 hours at a sound level of 80 dBA to 0.25 hours at115 dBA. For every 5 dB increase in the noise level, the allowableexposure duration is halved. Non-industrial, e.g., recreational, noisesintense enough to damage hearing can vary, for example, fromapproximately 90 dBA (lawnmower) to approximately 140 dBA (shotgunblast).

Radiation

Exposure to radiation, whether intentional, as in radiation therapy, orunintentional, as by accident, war, etc., can result in ototoxicity, aswell as neural damage (neurotoxicity), alopecia, gastrointestinaldisorders, and reduced patient survival. Although physical rather thanchemical, radiation can be considered another “ototoxin” in view of itstoxicity to the ear and hearing. Radiation-induced hearing loss is morelikely to involve the middle ear than is. hearing loss caused byplatinum-containing compounds or loop diuretics; however, cochlear andneural problems can also occur.

Radiation-induced ototoxicity, for example hearing loss, can occur as aresult of exposure to 35-40 Gy or higher, either as a single orcumulative dose. Radiation-induced gastrointestinal toxicity, which issimilar to that occurring during chemotherapy, includes electrolyteloss, secondary infections, bloody diarrhea, and gastrointestinalbleeding, and can occur upon exposure to a radiation dose in the rangefrom 5-20 Gy, or higher.

Administration of Methionine Protective Agents

The methionine protective agents of the present invention can beadministered orally or parenterally, for example intraperitoneally, byintravenous injection, intravenous infusion, etc., as described inRemington's Pharmaceutical Sciences, Fifteenth Edition, Mack PublishingCompany, Easton, Pa., 1975. These protective agents can also be given bylocal administration, for example, when the platinum-containingchemotherapeutic agent is administered by local injection, as notedabove. Localized administration of methionine protective agents can becarried out by topical application employing pharmaceutical formulationsdesigned for this purpose as is known in the art, local injection, etc.

In one embodiment of the present invention, a methionine protectiveagent is administered topically to the round window membrane of the ear.Typically, such topical administration is carried out by applying apharmaceutical formulation such as a topical solution comprising theotoprotective agent to the round window membrane by a micro-catheter orby injection into the middle ear. Suitable micro-catheters include thosecommercially available, for example, from IntraEar Corp., Denver, Colo.Preferably, the micro-catheter is attached to a battery-operated pumpsuch as that commercially available from Disetronics, Inc., which iscapable of automatically applying a topical solution comprising theotoprotective agent to the round window membrane, either continuously orintermittently.

Administration of the methionine protective agents of the presentinvention simultaneously with the administration of aplatinum-containing chemotherapeutic agent, loop diuretic agent,aminoglycoside antibiotic, iron chelating agent, or quinine orquinidine, can be accomplished in several ways. For example, each agentcan be formulated individually and administered separately at the sametime via any of the routes described herein or which are otherwiseconventional in the art. Alternatively, both can be contained togetherin a single dose formulation that can be administered by a single route.As in the case of the platinum-containing chemotherapeutic agent, loopdiuretic agent, etc., the dose of methionine protective agent can beadministered in a single day.

Dosages

The methionine protective agents discussed above can be employed inmethods for treating human and animal patients undergoing treatment withanti-cancer effective amounts of platinum-containing chemotherapeuticagents to prevent or reduce ototoxicity, weight loss, gastrointestinaltoxicity, neurotoxicity, alopecia, and to prolong survival. In addition,these methionine protective agents can be employed in methods fortreating human and animal patients undergoing treatment withdiuretic-effective amounts of loop diuretic agents, antibiotic-effectiveamounts of aminoglycoside antibiotics, iron chelating-effective amountsof iron chelating agents, effective amounts of quinine or quinidine, orin methods for treating human and animal patients exposed to ototoxicnoise levels, and methods for treating human and animal patients exposedto radiation levels capable of causing ototoxic effects such as hearingloss, as well as radiation-induced neural damage, alopecia, andgastrointestinal disorders. The present methionine protective agents canalso improve survival in patients exposed to radiation.

These methods comprise administering to the patient an appropriateeffective amount of a methionine protective agent prior to,simultaneously with, or subsequent to administration of aplatinum-containing chemotherapeutic agent, loop diuretic agent,aminoglycoside antibiotic, iron chelating agent, or quinine orquinidine, or exposure of the patient to noise or radiation.Combinations of these time periods can also be employed.

Typically, the methionine protective agents of the present invention canbe administered orally; parenterally by intravenous injection or slowinfusion; intraperitoneally; or topically by application to the roundwindow membrane of the ear. When administered parenterally, theeffective amount of methionine protective agent can be in the range offrom about 1.0 mg/kg body weight to about 600 mg/kg body weight. Morepreferably, the effective amount of methionine protective agent rangesfrom about 5 mg/kg body weight to about 500 mg/kg body weight, even morepreferably from about 10 mg/kg body weight to about 400 mg/kg bodyweight.

Alternatively, the effective amount of methionine protective agent canbe expressed on a mole:mole basis in relation to the anti-cancereffective amount of platinum-containing chemotherapeutic agent. Thiseffective amount can be in the range of from about 4:1 to about 167:1,more preferably from about 4.25:1 to about 100:1, and most preferablyfrom about 4.68:1 to about 20:1, methionine protectiveagent:platinum-containing chemotherapeutic agent, on a molar basis. Adosing ratio of about 18.75:1 on a molar basis is a preferred ratio.

If necessary, the amounts and ratios described above can be modified fordifferent platinum-containing chemotherapeutic agents, loop diureticagents, aminoglycoside antibiotics, iron chelating agents, and quinineor quinidine, or exposure to noise and radiation, by routineoptimization, including monitoring of effectiveness and titration forthe desired effect, by the methods described herein.

When administered orally, the methionine protective agent should begiven in an amount that will result in a blood serum level equivalent tothat achieved by the parenterally administered dosages set forth above.Such effective oral dosages can easily be determined by one of ordinaryskill in the art via conventional in vitro or in vivo methods such asthose described in Remington's Pharmaceutical Sciences, FifteenthEdition, Mack Publishing Company, Easton, Pa., 1975.

When administered topically to the round window membrane of the ear, theeffective amount of otoprotective agent is typically administered as apharmaceutical formulation such as a topical solution. Generally,topical administration comprises applying from about 0.001 ml to about0.010 ml, preferably from about 0.0025 ml to about 0.0075 ml, and mostpreferably about 0.005 ml of a topical solution comprising theotoprotective agent to the round window membrane. The topical solutiontypically comprises from about 10 mg/ml to about 50 mg/ml, preferablyfrom about 20 mg/ml to about 30 mg/ml, and most preferably about 25mg/ml of otoprotective agent.

Treatment Regimen

In the various methods of the present invention, the effective amount ofsulfur-containing protective agent can be administered prior to,contemporaneously with, or subsequent to administration of the effectiveamount of platinum-containing chemotherapeutic agent, loop diureticagent, aminoglycoside antibiotic, iron chelating agent, or quinine orquinidine, or exposure of the patient to harmful noise or radiation.Combinations of these time periods can also be employed. Generally,prior administration of the effective amount of the otoprotective agentcan be conducted broadly within the period ranging from as much as 14days (i.e., about 336 hours, about 168 hours, about 84 hours or about 60hours or less) before administration of the platinum-containingchemotherapeutic agent, loop diuretic agent, etc., or exposure to noiseor radiation. Likewise, subsequent administration of the effectiveamount of the otoprotective agent can be conducted broadly within theperiod including as much as 14 days (i.e., including about 60 hours,about 84 hours, about 168 hours or about 336 hours or more) afteradministration of the platinum-containing chemotherapeutic agent, loopdiuretic agent, etc., or exposure to noise or radiation.

Preferably, prior administration of the effective amount of themethionine protective agent is within about 48 hours beforeadministration of the platinum-containing chemotherapeutic agent, loopdiuretic agent, etc., or exposure to noise or radiation; with subsequentadministration within about 48 hours after administration of theplatinum-containing chemotherapeutic agent, loop diuretic agent, etc.,or exposure to noise or radiation. More preferably, prior administrationcan be within about 36 hours before administration of theplatinum-containing chemotherapeutic agent, loop diuretic agent, etc.,or exposure to noise or radiation; and subsequent administration can bewithin about 36 hours after administration of the platinum-containingchemotherapeutic agent, loop diuretic agent, etc., or exposure to noiseor radiation. Still more preferably, prior administration can be withinabout 25 hours before, and subsequent administration can be within about25 hours after, administration of the platinum-containingchemotherapeutic agent, loop diuretic agent, etc., or exposure toharmful levels of noise or radiation. Even more preferably, prioradministration can be within about 6 hours before, and subsequentadministration can be within about 1 hour after, administration of theplatinum-containing chemotherapeutic agent, loop diuretic agent, etc.,or exposure to noise or radiation. Even more preferably, prioradministration of the effective amount of methionine protective agentcan be within about 1 hour before, and subsequent administration can bewithin about 1 hour after, administration of the platinum-containingchemotherapeutic agent, loop diuretic agent, etc., or exposure to noiseor radiation. Still more preferably, prior administration of theeffective amount of methionine protective agent can be within aboutone-half hour before, and subsequent administration can be within aboutone-half hour after, administration of the platinum-containingchemotherapeutic agent, loop diuretic agent, etc., or exposure to noiseor radiation.

The platinum-containing chemotherapeutic agent can be administeredparenterally, for example by slow intravenous infusion, or by localinjection, as discussed above. Loop diuretic agents can be administeredorally or parenterally, for example by slow intravenous infusion or bylocal injection, as is well known in the art. Aminoglycosideantibiotics, iron chelating agents, quinine, quinidine, and radiationcan also be administered by routes and in doses conventional in the art.The methionine protective agent can be administered orally, parenterallyby intravenous injection or slow infusion, intraperitoneally ortopically to the round window membrane.

In a preferred embodiment of the present invention, when treating orpreventing otoxicity due to exposure to noise, the effective amount ofthe otoprotective agent can be administered prior to, simultaneouslywith, or subsequently to the noise exposure. For example, it has beenfound that administering the otoprotective agent twice daily for aperiod ranging from about 14 days (i.e., from about 336, 168, 84, or 60hours or less) before the noise exposure to about 14 days (i.e., from upto about 60, 84, 168, or 336 hours) after the noise exposure, preferablyfrom about 7 days before the exposure to noise to about 7 days after theexposure to noise, and more preferably from about 48 hours before theexposure to noise to about 48 hours after the exposure to noise (i.e.,from about 36, 24, 12, 6, 4, 2, 1 or ½ hour before to about ½, 1, 2, 4,6, 12, 24 or 36 hours after the exposure to noise), can significantlyameliorate or prevent ototoxicity in a human or animal patient.

When the otoprotective agent is administered to the round windowmembrane, the otoprotective agent may be administered continuously orperiodically during any of the time periods described above byintroducing a topical solution into the ear as described above, i.e., byadministration with a micro-catheter or by injection into the middleear, preferably by administration with a micro-catheter equipped with anautomatic, battery-operated pump.

Delayed toxic effects due to platinum-containing chemotherapeutic agentshave been observed. The protective effects of the present methionineprotective agents can be enhanced by administering them in asupplemental manner during the course of the patient's chemotherapyand/or afterwards as necessary or as desired. The same considerationsapply in the case of loop diuretics, aminoglycoside antibiotics, ironchelating agents, quinine, quinidine, noise-induced hearing damage, andradiation exposure. Thus, the methods described herein can furthercomprise semi-daily, daily or weekly administration of a supplementalamount of methionine protective agent.

Stated another way, it is often beneficial to administer supplementaldoses of the otoprotective agents of the present invention so as tomaintain effective blood serum levels of the otoprotective agents.Generally, the administration of supplemental amounts of otoprotectiveagents should result in the blood serum level of the human or animalpatient being maintained within at least about 10%, preferably fromabout 20% to about 70%, and more preferably within about 40%, of theblood serum level of the patient that results from the administration ofthe effective amount of otoprotective agent. Typically, suchsupplemental doses are administered within the time frames and dosagesset forth above for the effective amount of otoprotective agents, forexample, semi-daily, daily or weekly for a period of from about one tofourteen days after the administration of the effective amount.

As with the effective amount of methionine protective agent describedabove, the supplemental methionine protective agent can be administeredorally; parenterally by intravenous injection or slow infusion;intraperitoneally or topically by application to the round windowmembrane. When administered parenterally, the supplemental amount of themethionine protective agent is preferably in the range of from about 1.0mg/kg body weight to about 600 mg/kg body weight, more preferably fromabout 5 mg/kg body weight to about 500 mg/kg body weight, even morepreferably from about 10 mg/kg body weight to about 400 mg/kg bodyweight.

Alternatively, the supplemental amount of methionine protective agentparenterally administered daily or weekly can be expressed on amole:mole basis in relation to the anti-cancer effective amount ofplatinum-containing chemotherapeutic agent. This effective amount can bein the range of from about 4:1 to about 167:1, more preferably fromabout 4.25:1 to about 100:1, and most preferably from about 4.68:1 toabout 20:1, methionine protective agent:platinum-containingchemotherapeutic agent, on a molar basis. A dosing ratio of about18.75:1 on a molar basis is preferred.

Oral or parenteral doses administered daily can be within the lowerranges listed above. When administered orally, daily or weekly dosesshould be designed to achieve serum levels equivalent to those achievedby administration of the various parenteral doses described above.

When administered topically to the round window membrane of the ear, thesupplemental amount of otoprotective agent may be administered in thesame way as described above for the effective amount, typically as apharmaceutical formulation such as a topical solution. Generally, thesupplemental topical administration comprises applying from about 0.001ml to about 0.010 ml, preferably from about 0.0025 ml to about 0.0075ml, and most preferably about 0.005 ml of a topical solution comprisingthe otoprotective agent to the round window membrane. The topicalsolution typically comprises from about 10 mg/ml to about 50 mg/ml,preferably from about 20 mg/ml to about 30 mg/ml, and most preferablyabout 25 mg/ml of otoprotective agent.

Optimization of Treatment Regimen

In the methods of preventing or reducing ototoxicity of the presentinvention, various parameters associated with the patient's hearing andvestibular systems can be tested by methods well known in the art toestablish pretreatment baseline values. After administration of themethionine protective agent, and over the course of chemotherapy andafterwards, ototoxic effects can be monitored by conventional tests, andthe results can be compared to those obtained prior to treatment todetermine if any change has occurred. If any impairment is observed, theamount and/or time of administration of the protective agentadministered in conjunction with subsequent doses of theplatinum-containing chemotherapeutic agent, loop diuretic agent,aminoglycoside antibiotic, iron chelating agent, quinine, quinidine, orexposure to noise or radiation, can be adjusted so as to reduce orprevent further ototoxic changes without substantially diminishing theantineoplastic effectiveness of the platinum-containing chemotherapeuticagent or radiation, the diuretic effect of the loop diuretic agent, etc.Similar modification of treatment parameters in the case of weight loss,gastrointestinal toxicity due to either the platinum-containingchemotherapeutic agent or radiation, neurotoxicity due to either theplatinum-containing chemotherapeutic agent or radiation, alopecia due toeither the platinum-containing chemotherapeutic agent or radiation, andoverall patient condition/survival due to either the platinum-containingchemotherapeutic agent or radiation can be employed to optimize theprotective effects of the protective agent with respect thereto. Thiscan be achieved via appropriate testing and comparison of pre- andpost-treatment values, e.g., patient weight and patientphysical/medical/physiological condition, etc., with protocoladjustments being made as needed.

The invention being thus described, it will be obvious that the same canbe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications and equivalents as would be obvious to one skilled inthe art are intended to be included within the scope of the followingclaims.

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1. A method for reducing ototoxicity in a patient in need thereofselected from the group consisting of a human, a cat, and a dogundergoing treatment with a chemotherapeutic effective amount of ananti-tumor platinum-coordination compound, the method comprisingadministering to said patient an effective amount of an otoprotectiveagent selected from the group consisting of L-methionine and a mixtureof D-methionine and L-methionine or a pharmaceutically acceptable saltsthereof.
 2. The method of claim 1, wherein said otoprotective agent isL-methionine.
 3. The method of claim 1, wherein said otoprotective agentis a mixture of D-methionine and L-methionine.
 4. The method of claim 1,wherein said otoprotective agent is administered prior to theadministration of said anti-tumor platinum-coordination compound.
 5. Themethod of claim 1, wherein said otoprotective agent is administeredsimultaneously with the administration of said anti-tumorplatinum-coordination compound.
 6. The method of claim 1, wherein saidotoprotective agent is administered subsequently to administration ofsaid anti-tumor platinum-coordination compound.
 7. The method of claim1, wherein said effective amount of said otoprotective agent isadministered to said patient in a time period from about 36 hours beforeadministration of said anti-tumor platinum-coordination compound toabout 36 hours after administration of said anti-tumorplatinum-coordination compound.
 8. The method of claim 1, wherein saideffective amount of said otoprotective agent is administered to saidpatient in a time period from about 25 hours before administration ofsaid anti-tumor platinum-coordination compound to about 25 hours afteradministration of said anti-tumor platinum-coordination compound.
 9. Themethod of claim 1, wherein said effective amount of said otoprotectiveagent is administered to said patient in a time period from about 6hours before administration of said anti-tumor platinum-coordinationcompound to about 6 hours after administration of said anti-tumorplatinum-coordination compound.
 10. The method of claim 1, wherein saideffective amount of said otoprotective agent is administered to saidpatient in a time period from about 1 hour before administration of saidanti-tumor platinum-coordination compound to about 1 hour afteradministration of said anti-tumor platinum-coordination compound. 11.The method of claim 1, wherein said effective amount of saidotoprotective agent is administered to said patient in a time periodfrom about one-half hour before administration of said anti-tumorplatinum-coordination compound to about one-half hour afteradministration of said anti-tumor platinum-coordination compound. 12.The method of claim 1, wherein said anti-tumor platinum-coordinationcompound is selected from the group consisting ofcis-diaminedichloroplatinum(II), trans-diaminodichloroplatinum(II),cis-diamine-diaquaplatinum(II)-ion,chloro(diethyl-enetriamine)-platinum(II) chloride,dichloro(ethylene-diamine)-platinum(II),diamine(1,1-cyclobutanedi-carboxylato)-platinum(II), spiroplatin,dichiorotrans-dihydroxybisisopropolamine platinum IV (iproplatin),diamine(2-ethylmalonato)-platinum(II),ethylenediamine-malonatoplatinum(II),aqua(1,2-diaminodyclohexane)-sulfatoplatinum(II),(1,2-diaminocyclohexane)malonato-platinum(II),(4-carboxy-phthalato)(1,2-diaminocyclo-hexane)-platinum(II),(1,2-diaminocyclohexane)-(isocitrato)platinum(II),(1,2-diaminocyclohexane)-cis(pyruvato)platinum(II), and(1,2-diaminocyclohexane)-oxalatoplatinum(II).
 13. The method of claim12, wherein said anti-tumor platinum-coordination compound comprisescis-diaminedichloro-platinum(II).
 14. The method of claim 1, whereinsaid anti-tumor platinum-coordination compound is selected from thegroup consisting of cisplatin, carboplatin and iproplatin.
 15. Themethod of claim 1, wherein said otoprotective agent is administeredparenterally, orally, or topically to the round window membrane of saidpatient.
 16. The method of claim 15, wherein the administration of saideffective amount of said otoprotective agent results in a blood serumlevel equivalent to that achieved by parenteral administration in therange of from about 0.1 mg/kg body weight to about 500 mg/kg bodyweight.
 17. The method of claim 15, wherein the administration of saideffective amount of said otoprotective agent results in a blood serumlevel equivalent to that achieved by parenteral administration in therange of from about 1 mg/kg body weight to about 400 mg/kg body weight.18. The method of claim 15, wherein the administration of said effectiveamount of said otoprotective agent results in a blood serum levelequivalent to that achieved by parenteral administration in the range offrom about 10 mg/kg body weight to about 300 mg/kg body weight.
 19. Themethod of claim 15, wherein the administration of said effective amountof said otoprotective agent results in a blood serum level equivalent tothat achieved by parenteral administration in the range of from about 10mg/kg body weight to about 75 mg/kg body weight.
 20. The method of claim1, wherein the molar ratio of the effective amount of said otoprotectiveagent to the effective amount of said anti-tumor platinum-coordinationcompound is from about 4:1 to about 167:1, otoprotectiveagent:platinum-coordination compound.
 21. The method of claim 1, whereinthe molar ratio of the effective amount of said otoprotective agent tothe effective amount of said anti-tumor platinum-coordination compoundis from about 4.25:1 to about 100:1, otoprotectiveagent:platinum-coordination compound.
 22. The method of claim 1, whereinthe molar ratio of the effective amount of said otoprotective agent tothe effective amount of said anti-tumor platinum-coordination compoundis from about 4.68:1 to about 20:1, otoprotectiveagent:platinum-coordination compound.
 23. The method of claim 1, whereinthe molar ratio of the effective amount of said otoprotective agent tothe effective amount of said anti-tumor platinum-coordination compoundis about 18.75:1, otoprotective agent:platinum-coordination compound.24. The method of claim 1, further comprising administering to saidpatient a supplemental amount of said otoprotective agent during and/orafter the course of treatment with said anti-tumor platinum-coordinationcompound.
 25. The method of claim 24, wherein the supplemental amount ofsaid otoprotective agent is administered orally, parenterally, ortopically.
 26. The method of claim 25, wherein the administration ofsaid supplemental amount of said otoprotective agent is sufficient tomaintain an effective blood serum level of the otoprotective agent insaid patient for a period of from one to fourteen days during and/orafter the administration of said anti-tumor platinum-coordinationcompound.
 27. The method of claim 26, wherein the administration of saidsupplemental amount of said otoprotective agent results in a blood serumlevel equivalent to that achieved by parenteral administration in therange of from about 0.1 mg/kg body weight to about 500 mg/kg body weightper week during and/or after the course of treatment with saidanti-tumor platinum-coordination compound.
 28. A method for reducingototoxicity in a patient selected from the group consisting of a human,a cat, and a dog undergoing treatment with a chemotherapeutic effectiveamount of an anti-tumor platinum-coordination compound, the methodcomprising administering to said patient an effective amount of anotoprotective agent comprising L-methionine, D,L-methionine or apharmaceutically acceptable salt thereof, the administration of saideffective amount of said otoprotective agent resulting in a blood serumlevel equivalent to that achieved by parenteral administration in therange of from about 1 mg/kg body weight to about 100 mg/kg body weight.29. The method of claim 28, further comprising administering to saidpatient a supplemental amount of said otoprotective agent, theadministration of said supplemental amount resulting in a blood serumlevel equivalent to that achieved by parenteral administration in therange of from about 0.1 mg/kg body weight to about 500 mg/kg body weightper week.